Incorporation of FeoB into synthetic lipid vesicles enables functional GTPase activity in a cell-free artificial membrane system

<ns5:p> Background Ferrous iron uptake is essential for the survival and virulence of many bacterial pathogens. The inner membrane protein FeoB is a key component of iron import, coupling ferrous iron transport to GTP hydrolysis through conserved G-protein domains. Despite the importance of this protein, studying FeoB in isolation remains challenging, as FeoB is an integral membrane protein whose function and stability depend on lipid membrane integration. Methods Full-length <ns5:italic>E. coli</ns5:italic> FeoB constructs fused to YFP were engineered and expressed in vitro. Purified FeoB was reconstituted into giant unilamellar vesicles composed of 1:2 POPC:POPG using a water-in-oil emulsion–transfer method. Bioinformatic analyses validated the domain architecture of FeoB. We assessed <ns5:italic>in vitro</ns5:italic> expression with both homemade cell-free extracts and the PURE system, evaluated protein encapsulation by confocal microscopy, and quantified GTPase activity using a luminescence-based assay under anaerobic conditions. Results FeoB-YFP was successfully expressed and purified, with expression enhanced at lower temperatures. Bioinformatic analysis confirmed the presence of conserved G-protein motifs and transmembrane domains. Encapsulation of FeoB within synthetic vesicles resulted in robust YFP fluorescence, indicating successful membrane integration. Vesicle-encapsulated FeoB bound a fluorescent GTP analog, demonstrating active G-domain function. Luminescence-based assays revealed FeoB GTPase activity that was potentiated by ferrous iron and further enhanced by potassium ions. Conclusions We reconstituted and functionally characterized FeoB within controlled artificial membrane environments. The demonstration of active GTPase domains in reconstituted FeoB provides a foundation for dissecting iron transport mechanisms and for screening inhibitors targeting bacterial FeoB-mediated virulence pathways. </ns5:p>